Orientational aspects of antibody immobilization and immunological activity on quartz crystal microbalance electrodes

The resonant frequency shift of a quartz crystal microbalance (QCM) has been used to monitor the immobilization of immunoglobulin G (IgG) and anti-IgG antibodies onto unmodified and modified gold QCM electrodes, IgG and anti-IgG antibodies have been immobilized onto, the gold QCM surfaces by four separate methods. First, the antibodies were immobilized directly onto unmodified gold QCM surfaces by physical adsorption. Second, 12-mercaptododecan-1-ol was reacted with the gold surface, and after reaction with bromoacetic acid, the antibodies were coupled to the thiolated surface using a water soluble carbodiimide hydrochloride and N-hydroxysuccinimide. Third, the antibodies were modified by the direct attachment of thiol groups using 2-iminothiolane and subsequently immobilized onto the Bold surface. Fourth, the gold QCM surface was modified by the direct adsorption of Protein A, followed by antibody adsorption. Physical adsorption of the antibodies onto gold QCM electrodes resulted in 30% more bound antibody compared with the covalent attachment and Protein A immobilization methods, Atomic force microscopy( AFM) images of adsorbed IgG onto gold-coated mica revealed the antibodies to be aggregated and randomly distributed on the surface, Tile immobilized receptor layers, prepared by the above four procedures, were allowed to interact with their biospecific partner species, The resulting QCM frequency shifts were used to evaluate both the orientational aspects of antibody immobilization and immunological activities of the immobilized layers. Receptor layers formed by the immobilization of antibodies onto thiolated QCM electrodes did not increase the amount of antibody subsequently bound compared with receptor layers prepared by direct physical adsorption of antibodies, Receptor layers of thiolated antibodies yielded varied immunological activities, The protein A coupling layer between the antibody receptor layer and gold QCM surface facilitated subsequent antibody binding, (C) 1996 Academic Press, Inc.